喉鳞癌患者外周血CD45low CD34+ KDR+内皮祖细胞的检测及其临床意义

目的:研究喉鳞癌患者外周血中循环CD45lowCD34+KDR+内皮祖细胞(endothelial progenitor cells,EPCs)的水平及其与喉鳞癌发生、发展的相关性。方法:收集2010年2月至2011年2月20例喉鳞癌患者外周血标本,10例健康志愿者外周血作为对照。流式细胞术检测CD45lowCD34+KDR+EPCs占外周血CD34+造血干细胞或外周血淋巴细胞的比例,实时定量PCR检测PBMC中KDR mRNA和CD133 mRNA的表达水平,ELISA与Griess法分别检测血清中VEGF和NO的水平。结果:临床Ⅱ期~Ⅳ期的喉鳞癌患者外周血CD45lowCD34+KDR+EPCs占CD34+造血干细胞的比例和占淋巴细胞的比例均较健康对照组明显升高[(55.4±11.4)%vs(21.0±5.8)%,P<0.05;(1.27±0.25)vs(0.25±0.09),P<0.05]。临床Ⅱ期~Ⅳ期喉鳞癌患者PBMC中KDR mRNA和CD133 mRNA的表达水平及血清中VEGF水平均明显高于健康对照组(P<0.05),NO水平没有明显差异;而临床Ⅰ期的喉鳞癌患者CD45lowCD34+KDR+EPCs比例、KDR mRNA和CD133 mRNA表达以及VEGF水平与健康对照组相比无差异。结论:晚期喉鳞癌患者外周血循环CD45lowCD34+KDR+EPC和血清中VEGF水平均明显升高,可能与肿瘤的发生有关。     

喉鳞癌患者外周血CD45~(low)CD34~+KDR~+内皮祖细胞的检测及其临床意义

目的:研究喉鳞癌患者外周血中循环CD45lowCD34+KDR+内皮祖细胞(endothelial progenitor cells,EPCs)的水平及其与喉鳞癌发生、发展的相关性。方法:收集2010年2月至2011年2月20例喉鳞癌患者外周血标本,10例健康志愿者外周血作为对照。流式细胞术检测CD45lowCD34+KDR+EPCs占外周血CD34+造血干细胞或外周血淋巴细胞的比例,实时定量PCR检测PBMC中KDR mRNA和CD133 mRNA的表达水平,ELISA与Griess法分别检测血清中VEGF和NO的水平。结果:临床Ⅱ期~Ⅳ期的喉鳞癌患者外周血CD45lowCD34+KDR+EPCs占CD34+造血干细胞的比例和占淋巴细胞的比例均较健康对照组明显升高[(55.4±11.4)%vs(21.0±5.8)%,P<0.05;(1.27±0.25)vs(0.25±0.09),P<0.05]。临床Ⅱ期~Ⅳ期喉鳞癌患者PBMC中KDR mRNA和CD133 mRNA的表达水平及血清中VEGF水平均明显高于健康对照组(P<0.05),NO水平没有明显差异;而临床Ⅰ期的喉鳞癌患者CD45lowCD34+KDR+...     

乳腺癌患者外周血中内皮祖细胞检测的临床意义

目的探讨乳腺癌患者外周血中内皮祖细胞(EPCs)检测的临床意义。 方法采用前瞻性研究方法,收集2014年3月至2015年3月吉林省肿瘤医院收治的、病理学证实的70例浸润性乳腺导管癌患者、61例乳腺纤维腺瘤患者和68名健康人的外周血,利用流式细胞术检测EPCs[CD34、CD133和血管内皮生长因子受体(VEGFR)-2阳性细胞]水平。采用Fisher确切概率检验和Kruskal-Wallis H检验比较3组间EPCs阳性率及其表面标志物CD34、CD133和VEGFR-2表达量的差异,并用Fisher确切概率检验分析乳腺癌患者EPCs阳性率与临床病理特征的关系;采用t检验比较不同临床分期和不同淋巴结转移状态患者间EPCs表面标志物CD34、CD133和VEGFR-2表达量的差异。 结果在乳腺癌患者、乳腺纤维腺瘤患者和健康人外周血中,EPCs阳性率及其表面标志物CD34、CD133和VEGFR-2表达量的差异均有统计学意义(χ²＝12.811,P<0.001;F＝15.275,P<0.001);健康人及乳腺纤维腺瘤组均未检测到EPCs,组间两两比较显示,EPCs表面标志物CD34、CD133和VEGFR-2在乳腺癌患者外周血中的表达量[M(P₂₅～P₇₅)：0.006%(0.003%～6.008%)]明显高于健康人及乳腺纤维腺瘤患者(P＝0.002、0.003),乳腺癌组EPCs阳性率也显著高于健康人及乳腺纤维腺瘤患者[11.4%(8/70)分别比0(0/68)、0(0/61),P＝0.006、0.007]。但EPCs阳性率与乳腺癌患者的年龄、ER、PR和HER-2状态均无关(P均>0.050)。进一步分析EPCs阳性的8例乳腺癌患者临床资料后发现,Ⅰ期患者(n＝4)外周血中EPCs表面标志物CD34、CD133和VEGFR-2的表达量明显低于Ⅱ～Ⅳ期患者(n＝4)[(0.300±0.162)%比(1.130±0.318)%,t＝4.640,P＝0.004],而淋巴结转移者(n＝4)EPCs表面标志物CD34、CD133和VEGFR-2的表达量明显高于未转移者(n＝4)[(1.062±0.424)%比(0.370±0.287)%,t＝2.700,P＝0.040]。 结论乳腺癌患者外周血中EPCs表面标志CD34、CD133和VEGFR-2表达水平较高,其可能是一种潜在的生物标志物和治疗靶点。     

流式细胞术检测结直肠癌根治术前后循环肿瘤细胞和循环肿瘤干细胞及其临床预测价值

目的:探讨流式细胞仪检测结直肠癌根治术前后循环肿瘤细胞(circulating tumor cell,CTC)和循环肿瘤干细胞(circulating tumor stem cell,CTSC)作为患者临床预测指标的可行性和临床价值.方法:人组首诊初治50例结直肠癌患者,手术前后各抽取15 ml外周血.分离单个核细胞后分成两份,一份标记CTC标志物(CD45、EpCAM和CK),另一份标记CTSC标志物(CD45、EpCAM、CD44和CD133),Moflo XDP流式细胞仪对其进行检测,CD45-EpCAM+ CK+细胞确定为CTC;CTSC确定为3群:CD44+CTSC、CD133+ CTSC和CD44+ CD133+ CTSC.结果:结直肠癌根治术前患者CTC和CD44+ CTSC阳性率分别为34％和44％,CD133+ CTSC和CD44+CD133+ CTSC是24％和0;术后患者CTC和CD44+ CTSC阳性率分别为38％和54％,CD133+ CTSC和CD44+ CD133+ CTSC分别为26％和0.根治术前后CTC阳性率都与肿瘤T分期有关联(P＜0.05);术后CTC与肿瘤的浸润深度、淋巴结转移和远处转移有关联(P＜0.05);术前CD44+ CTSC与肿瘤的浸润深度有关联.结论:根治术前后CTC及术前CD44+ CTSC阳性率都具有临床预测指标的可行性和价值,术后CTC阳性率可能是更好的预测指标.     

肝细胞性肝癌组织CD133+细胞肿瘤干细胞特性的研究

目的 越来越多的研究表明,肝癌细胞系中能检测到肿瘤干细胞(cancer stem cells,CSCs)的存在.本研究旨在探讨从肝细胞性肝癌(hepatocellular carcinoma,HCC)组织样本中分离获得的CD133+细胞是否具有CSCs特性.方法 将2014-02-01-2015-06-30广西医科大学附属肿瘤医院肝胆外科25例手术获得的新鲜HCC组织和对应癌旁组织,采用酶消化法分别消化成单个肝癌细胞和单个肝细胞,利用流式细胞术检测部分单个肝癌细胞和单个肝细胞CD133的表达率.用剩余的单个肝癌细胞进行原代培养,流式细胞术将培养获得的肝癌细胞分选为CD133+和CD133-细胞,通过平板克隆形成实验、肿瘤球形成实验和裸鼠移植瘤形成实验对比分析这两组细胞的CSCs特性.结果 25例HCC组织中CD133的表达率为3.8％～8.3％,平均值为(5.8±1.6)％,而癌旁组织CD133的表达率为0.1％～0.4％,平均值为(0.2±0.1)％,两者比较差异有统计学意义,t=17.12,P＜0.001.CD133+和CD133-细胞的平均克隆率分别为(25.2±0.8)％和(7.6±0.8)％,两者比较差异有统计学意义,t=81.95,P＜0.001.CD133+和CD133-细胞的平均成球率分别为(20.3±0.6)％和(12.5±1.4)％,两者比较差异有统计学意义,t=68.17,P＜0.001.CD133+细胞的裸鼠移植瘤形成能力明显高于CD133-细胞.结论 从HCC组织样本中分离获得的CD133+细胞具有明显的CSCs特性.     

内皮祖细胞诱导培养及携带基因腺病毒转染后生物活性变化的观察

目的:研究用人骨髓诱导培养内皮祖细胞(EPCs)的方法,探讨携带目的基因增殖缺陷的腺病毒对EPCs的转染情况、目的基因的表达情况和转染前后EPCs生物活性的变化.方法:用梯度离心法从人骨髓分离单个核细胞,加入人血管内皮生长因子(VEGF165)诱导培养,用流式细胞仪测定细胞表面标志CD14、CD34、CD45、CD166、CD29、CD44、HLA-DR、CD31、CD106、CD133和VEGFR2(KDR).增强型绿色荧光蛋白基因(EGFP)作为报告基因,流式细胞仪检测腺病毒载体(Ad-EGFP)对EPCs的感染效率.用携带人干扰素γ(hγIFN)基因的腺病毒载体(Ad-hγIFN)感染EPCs(EPCs-hγIFN),ELISA检测细胞EPCs-hγIFN分泌hγIFN的情况.MTT实验观察EPCs、EPCs-EGFP和EPCs-hγIFN的增殖情况,流式细胞仪检测EPCs、EPCs-EGFP和EPCs-hγIFN细胞CD133、KDR和CD34的表达情况.结果:从骨髓分离的单核细胞经过诱导培养2周后,贴壁的单核细胞开始表达EPCs标志CD133、KDR和CD34.Ad-EGFP感染复数(MOI)为150 pfu/EPCs时,感染率即可达90％以上.EPCs-hγIFN培养上清可以测得较高浓度而且浓度稳定的hγIFN.腺病毒转染后,EPCs的增殖活性和表面标志表达无显著性变化.结论:从人骨髓可以培养出EPCs,腺病毒可以把目标基因成功转入EPCs,转入的基因可以稳定地表达,在携带目的基因的腺病毒转染后,EPCs的细胞生物活性无显著性改变.     

原发性肝癌患者调节性T细胞与预后的关系

目的:探讨肝细胞肝癌(HCC)患者T淋巴细胞亚群对其预后的影响.方法:对138例HCC患者的临床病理资料进行回顾性分析,采用流式细胞技术(FCM)检测早晚期HCC患者外周血T淋巴细胞不同分群占外周血单个核细胞(PBMC)的比例,Kaplan-Meier法检验T淋巴细胞亚群对HCC患者生存的影响.结果:138例HCC患者的1年生存率为61%,2年生存率为39%,早晚期HCC患者的中位生存时间分别为28、12个月.晚期HCC患者的CD4+T细胞比例低于早期患者,调节性T细胞(Treg)与NK细胞比例高于早期患者,并且差异均有统计学意义(P<0.05).单因素分析显示T淋巴细胞亚群中Treg是影响HCC患者预后的相关因素.在进一步分析影响Treg的因素时发现有肝硬化的HCC患者外周血Treg比例及绝对数高于无肝硬化的HCC患者(P=0.029),铁蛋白(Ferr)正常的HCC患者外周Treg绝对数高于Ferr水平高的HCC患者(P=0.027).结论:HCC患者的Treg在一定程度上影响其预后,Treg比例>7%是影响晚期肝硬化HCC患者预后的独立危险因素.     

FL联合TPO体外培养AC133+细胞表面标记的变化

目的:探讨脐血造血干/祖细胞的体外扩增.方法:免疫磁珠法分离纯化脐血AC133+细胞,在细胞因子FLT3配体、血小板生成素作用下,体外扩增,检测有核细胞扩增的倍数.采用流式细胞仪分析细胞表面标志的变化.结果:FLT3联合血小板生成素体外培养2周.有核细胞扩增(18±8)倍.CD34细胞扩增2.8倍,AC133+未获扩增,CD34细胞纯度由(56±23)%下降到(8±1)%,AC133+细胞由85%下降到(0.1±0.1)%.随着体外培养时间延长至4周,有核细胞,CD34+进一步扩增.分别扩增475倍和17倍.但细胞随之发生分化,CD34+细胞占有核细胞中的比例下降至2%,AC133+细胞消失.CD45细胞上升到1.3%.结论:FL联合TPO长期培养AC133+细胞能使CD34细胞扩增.     

DC-CIK对急性髓细胞白血病干细胞的杀伤作用

目的:研究树突状细胞(dendritic cell,DC)联合同源细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对急性髓细胞白血病细胞株KG-1a中白血病干细胞(leukemic stem cell,LSC)的体外杀伤和诱导凋亡作用。方法:分离健康人外周血单个核细胞,贴壁细胞用GM-CSF和IL-4诱导培养DC,悬浮细胞用IL-2、IL-1、IFN-γ和CD3 mAb诱导培养CIK。将KG-1a细胞冻融物作为抗原负载DC(即Ag-DC),与CIK共培养作为实验组(Ag-DC-CIK),无抗原负载的DC与CIK共培养作为对照组(DC-CIK),单独CIK作为空白对照组,与KG-1a共育后流式细胞术检测各组细胞中CD34+CD38-CD123+白血病干细胞的比例。DC-CIK与KG-1a细胞共培养,流式细胞术检测各组细胞中KG-1a细胞与CD34+CD38-CD123+细胞的凋亡率。结果:外周血单个核细胞成功诱导DC。CIK组、DC-CIK组及Ag-DC-CIK组中CD3+CD56+细胞比例为(17.36±4.44)%、(28.22±3.66)%和(36.16±5.88)%,依次升高(P<0.05)。与对照组相比,Ag-DC-CIK组与DC-CIK组细胞中CD34+CD38-CD123+细胞比例显著降低[(8.78±0.62)%vs(3.95±0.53)%、(3.03±0.62)%,P<0.01〗。DC-CIK可诱导KG-1a细胞凋亡,凋亡率由(2.34±0.74)%上升至(12.27±1.01)%,但对其中CD34+CD38-CD123+细胞无明显的诱导凋亡作用。结论:DC联合CIK能杀伤急性髓细胞白血病干细胞,但无明显的诱导凋亡作用。     

调节性T细胞在慢性粒细胞白血病中的表达及临床意义

  目的   分析CD4+CD25+Treg细胞在慢性粒细胞白血病（CML）患者不同病程中的表达,探讨其与该疾病的疗效及预后的关系,从免疫学方面揭示CML发生及转归的可能机制。   方法   收集23例不同病程分期CML患者及10例健康对照的外周血,采用流式细胞仪技术对CD4+CD25+Treg细胞进行检测,比较CML患者与对照之间及不同病程CML患者之间CD4+CD25+Treg细胞表达的差异;比较CD4+CD25+Treg细胞表达水平与对应的临床参数间的关系。   结果   与正常对照比较,CML初治患者、加速急变期患者外周血单个核细胞中CD4+CD25+Treg细胞百分比呈显著性下降（P < 0.05）;而CML慢性治疗期患者外周血单个核细胞中CD4+ CD25+Treg细胞百分比与正常对照比较无显著性差异（P>0.05）;与正常对照比较,CML初治患者、加速急变期患者外周血单个核细胞中CD4+Treg细胞百分比呈显著性下降（P < 0.05）;而CML慢性治疗期患者外周血单个核细胞中CD4+Treg细胞百分比与正常对照比较无显著性差异（P>0.05）;不同病程CML患者外周血单个核细胞中CD4+CD25+Treg细胞百分比相比较,慢性稳定期患者较初治患者有明显上升,差异有统计学意义（P < 0.05）;而其他组之间无显著性差异（P>0.05）;初治CML患者外周血单个核细胞中CD4+ CD25+Treg细胞百分比与外周血血红蛋白量呈正相关（P=0.010）;与骨髓中幼稚细胞（原始粒细胞+早幼粒细胞）百分比呈负相关（P=0.022）。   结论   CML患者外周血单个核细胞中CD4+CD25+Treg细胞占外周血单个核细胞的比例下降,提示患者进入加速急变期CD4+CD25+Treg细胞在CML患者中起到一定的免疫调节作用。      

Associations Between Infiltrating Lymphocyte Subsets and Hepatocellular Carcinoma

Aims: We aimed to analyze the phenotype of tumor-infiltrating lymphocytes (TILs) and non-tumor infiltrating lymphocytes (NILs) in HCC and non-tumor tissues, and evaluate relationships between changes in these cells and the prognosis of HCC. Methods: Lymphocytes were isolated from HCC and corresponding non-tumor tissues and tested by flow cytometry. For comparison, clinical parameters were analyzed. Results: Compared with the non-tumor tissue, tumor tissue had a lower intensity of NK, NKT andCD8+T cell infiltration. TILs had higher intensity of CD4+CD25+Foxp3+regulatory T cell (Treg cells) infiltration compared with that in NILs. The prevalence of Treg cells was associated with fewer CD8 + T lymphocytes in the HCC immune microenvironment. The frequencies of NK cells and CD8+T cells in TILs of HCC patients with metastasis less than 12 months were lower than those without metastasis. However, the frequency of Treg cells was higher than those without metastasis. Conclusion: These results suggest that the frequencies of CD8+T, NK and NKT cells as well as Treg cells in the tumor tissue of HCC are significantly associated with patient survival, and could be applied as predictive indicators for HCC prognosis.     

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.     

Expression of Fas antigen (CD95) in peripheral blood lymphocytes and in liver-infiltrating, cytotoxic lymphocytes in patients with hepatocellular carcinoma

BACKGROUND: Fas-expressing cytotoxic T lymphocytes (CTLs) are important antitumor immune effector cells in patients with hepatocellular carcinoma (HCC). The role of transforming growth factor beta 1 (TGF-beta1) in modulating the expression of Fas by CTLs is not known in HCC. The objectives of this study were to characterize the expression of Fas by CTLs and natural killer (NK) cells among peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) in patients with HCC and to correlate the association, if any, with serum TGF-beta1 levels. METHODS: PBLs from 18 patients with HCC and TILs from 5 HCC liver specimens were isolated, and Fas expression was analyzed by three-color flow cytometry. The results were compared with results from normal control volunteers (n = 19 individuals). Serum TGF-beta1 levels in patients with HCC were measured by enzyme-linked immunosorbent assay. RESULTS: The median percentage of Fas expression by CD3 positive T cells was significantly higher in patients with HCC compared with normal controls (54.37% vs. 32.03%, respectively; P = 0.0036), and this was attributable solely to Fas expression by CD4 positive PBLs (54.46% vs. 34.90%, respectively; P = 0.0234). In contrast, Fas expression was significantly higher in all the subtypes of TILs (CD3 positive, CD4 positive, CD8 positive, NK cells, and natural T cells) compared with controls (all P values were < 0.001). Tumor size was inversely proportional to the TGF-beta1 levels (correlation coefficient [r] = -0.725; P < 0.0001), which were correlated inversely with Fas expression by CD4 positive PBLs (r = -0.516; P = 0.01). CONCLUSIONS: In patients with HCC, TILs exhibit significantly increased expression of Fas compared with PBLs that may enhance their susceptibility to apoptotic mechanisms. Larger tumors were associated with lower serum TGFbeta1 levels, and this was correlated with greater Fas expression by CD4 positive PBLs.     

原发性肝癌患者CD4+CD25+CD127low调节性T细胞的检测及意义

探讨CD4+CD25+CD127low调节性T细胞（Tregs）在原发性肝细胞性肝癌（HCC）患者外周血中的变化及其临床意义。方法:采集40例乙型肝炎病毒（HBV）相关的HCC患者［巴塞罗那临床肝癌（BCLC）分期A期患者7例、B期患者8例、C期患者20例、D期患者5例］、35例慢性乙型肝炎（CHB）患者及28例正常健康人的外周抗凝血,应用CD4（PE-CY5）、CD25（FITC）、CD127（PE）三种特异性荧光抗体标记后,通过流式细胞术对CD4+CD25+CD127lowTregs水平进行三色荧光抗体检测。结果:HCC患者外周血CD4+CD25+CD127lowTregs占CD4+T细胞的百分比显著高于正常健康人（P<0.001）和CHB患者（P=0.017）,CHB患者外周血CD4+CD25+CD127lowTregs占CD4+T细胞的百分比高于正常健康人（P=0.035）;HCC中BCLC分期为C期的患者外周血CD4+CD25+CD127lowTregs占CD4+T细胞的百分比显著高于A期患者（P=0.020）和B期患者（P=0.019）。结论:CD4+CD25+CD127lowTregs水平异常增高可能是HCC免疫逃逸的一个重要机制,且其变化水平与临床病情的进展存在一定的相关性。      

CD44v6对实验性大肠癌肝转移的影响

目的：研究ＣＤ４４ｖ６在大肠癌肝转移中的作用，探索一种有效的抗肿瘤转移方法。方法：应用大肠癌肝转移模型来研究ＣＤ４４ｖ６单抗对大肠癌ＨＴ２９、Ｌｏｖｏ细胞裸小鼠实验性肝转移的影响，分析ＣＤ４４ｖ６单抗在抗大肠癌肝转移中的作用。结果：ＨＴ２９细胞对照组肝转移率为４／８，实验组肝转移率为２／８；Ｌｏｖｏ细胞对照组肝转移率为８／８，实验组肝转移率为０。结论：ＣＤ４４ｖ６单抗能有效阻断裸小鼠实验性大肠癌肝转移的发生。     

肝细胞性肝癌组织浸润淋巴细胞表型与分布研究

目的:肝细胞性肝癌组织浸润淋巴细胞与外周血T 细胞表型可能与肿瘤进展及预后相关,本研究检测肝癌患者组织及外周血T 细胞表型与分布,分析淋巴细胞表型变化与预后的关系。方法:分析2007年10月至12月中山医院147 例肝癌及癌旁组织浸润淋巴细胞表型（T 细胞或B 细胞表面标志物:CD3、CD8、CD4、CD20、CD19、Foxp 3）,表型与临床病理特征及预后的关系;检测26例肝癌外周血CD3、CD8、CD4 +T细胞数量并其比例变化。结果:癌巢内肿瘤浸润细胞明显少于癌周组织（P < 0.01）,癌周淋巴细胞主要分布于癌旁正常肝组织、汇管区,其与患者肝炎病史及肝硬化相关,表型以CD3 +T细胞为主,其中又以CD8 + 细胞毒性T 细胞为主;CD4 染色在多数病例为阴性,Foxp 3 仅在个别病例（15/ 109）呈阳性。肿瘤浸润淋巴细胞B 细胞标志CD20、CD19均为阴性。肿瘤组织内CD8 +T细胞浸润数量与预后正相关,而癌周浸润淋巴细胞数目与患者转移及复发无显著关系。结论:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织,肿瘤及癌周浸润细胞以CD8 + 细胞毒性T 细胞为主。肿瘤组织内CD8 +T细胞浸润数量与预后相关,而癌周浸润淋巴细胞数量与患者转移及复发无显著关系。      

Expression of Fas/FasL in CD8+ T and CD3+ Foxp3+ Treg Cells - Relationship with Apoptosis of Circulating CD8+ T Cells in Hepatocellular Carcinoma Patients

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, includinglymphocyte apoptosis. Several studies showed that Foxp3+T cells take part in inducing this process by expressingFasL in tumor patients. However, the relationship between apoptosis, CD8+T cells and Foxp3+T cells in HCCpatients is still unclear. The present study was designed to investigate the correlation between apoptosis levelsand Fas/FasL expression in CD8+T lymphocytes and Foxp3+T cells in patients with HCC. Methods: CD8+T cellsand CD3+Foxp3+T cells were tested from peripheral blood of HCC patients and normal controls and subjectedto multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas inCD8+T cells and FasL in CD3+Foxp3+T cells were evaluated. Serum TGF-β1 levels in patients with HCC weremeasured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, aswell as FasL expression in CD3+Foxp3+T cells was then evaluated. Results: The frequency of CD8+T cells bindingannexin V and Fas expression in CD8+T cells, were all higher in HCC patients than normal controls and theproportion of apoptotic CD8+T cells correlated with their Fas expression. Serum TGF-β1 levels correlated inverselywith CD3+Foxp3+T cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of CD8+T cellsand reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis inductionin Fas+CD8+T cells in vitro are required.     

High expression of CD44v9 and xCT in chemoresistant hepatocellular carcinoma: Potential targets by sulfasalazine

CD44v9 is expressed in cancer stem cells (CSC) and stabilizes the glutamate‐cystine transporter xCT on the cytoplasmic membrane, thereby decreasing intracellular levels of reactive oxygen species (ROS). This mechanism confers ROS resistance to CSC and CD44v9‐expressing cancer cells. The aims of the present study were to assess: (i) expression status of CD44v9 and xCT in hepatocellular carcinoma (HCC) tissues, including those derived from patients treated with hepatic arterial infusion chemoembolization (HAIC) therapy with cisplatin (CDDP); and (ii) whether combination of CDDP with sulfasalazine (SASP), an inhibitor of xCT, was more effective on tumor cells than CDDP alone by inducing ROS‐mediated apoptosis. Twenty non‐pretreated HCC tissues and 7 HCC tissues administered HAIC therapy with CDDP before surgical resection were subjected to immunohistochemistry analysis of CD44v9 and xCT expression. Human HCC cell lines HAK‐1A and HAK‐1B were used in this study; the latter was also used for xenograft experiments in nude mice to assess in vivo efficacy of combination treatment. CD44v9 positivity was significantly higher in HAIC‐treated tissues (5/7) than in non‐pretreated tissues (2/30), suggesting the involvement of CD44v9 in the resistance to HAIC. xCT was significantly expressed in poorly differentiated HCC tissues. Combination treatment effectively killed the CD44v9‐harboring HAK‐1B cells through ROS‐mediated apoptosis and significantly decreased xenografted tumor growth. In conclusion, the xCT inhibitor SASP augmented ROS‐mediated apoptosis in CDDP‐treated HCC cells, in which the CD44v9‐xCT system functioned. As CD44v9 is typically expressed in HAIC‐resistant HCC cells, combination treatment with SASP with CDDP may overcome such drug resistance.     

原发性肝癌经综合微创治疗后联合细胞因子诱导杀伤细胞灌注的近期疗效观察

背景与目的:近年来,微创治疗在原发性肝癌的临床应用中显示出良好的应用前景,而肿瘤免疫治疗也已成为肿瘤治疗研究的热点之一。本研究旨在探讨微创治疗联合细胞因子诱导的杀伤(cytokine-inducedkiller,CIK)细胞免疫治疗对防止肝癌复发的作用。方法:原发性肝癌患者85例(均为肝动脉化疗栓塞联合射频消融术后,经影像学检查显示病灶无活性且无远处转移),随机选45例为研究组,其余40例为对照组,研究组患者在微创治疗后联合CIK细胞经肝动脉回输,对照组患者不进行CIK细胞回输;CIK细胞治疗前及治疗结束后抽研究组患者外周血,采用流式细胞仪(FCM)测定T淋巴细胞亚群及NK细胞水平变化;研究组和对照组每2～3个月进行CT检查,了解肿瘤变化情况。结果:CIK细胞回输后CD3 、CD4 、CD56 (NK)效应细胞的比例和CD4 /CD8 比值显著上升,由最初的(68.6±11.0)%、(31.0±9.0)%、(15.6±7.8)%和1.1±0.5上升至(70.7±10.1)%(P<0.05)、(33.5±8.0)%(P<0.05)、(18.4±9.4)%(P<0.05)和1.3±0.6(P<0.05);CD8 和CD3 CD56 效应细胞比例下降,由回输前的(31.1±7.8)%和(6.4±3.5)%下降至回输后的(28.6±8.3)%(P<0.05)和(5.2±3.3)%(P<0.05)。研究组1年和18个月的复发率分别为8.89%和15.56%,而对照组为30.00%和40.00%(χ2值分别为4.87和6.41,P<0.05)。结论:微创治疗后联合CIK细胞免疫治疗可提高肝癌患者的免疫功能,对降低肝癌的复发率具有重要作用。